Liposomes encapsulating anticancer drugs and use thereof in the treatment of malignant tumours

ABSTRACT

Liposomes encapsulating anticancerous drugs and the use thereof in the treatment of malignant tumours. The liposomes are coated with a lipopeptide composed of three substructures: a lipid fragment, an active oligopeptide and an oligopeptide spacer between the other two fragments. Applicable in intravenous administration for treatment of malignant tumours.

FIELD OF THE INVENTION

The present invention is related with procuring a system ofanti-cancerous treatment capable of destroying selectively the cancerouscells inside a living being without affecting the remaining cells of thetreated organism. More particularly, the invention is related withliposomes containing anti-cancerous drugs of utility in theaforementioned treatment.

STATE OF THE TECHNIQUE

Cancer is one of the most widespread illnesses in developed countriesand, specifically, tumoral metastases are the main cause of mortality inpatients with solid malignant tumours. They consist of the appearance ofa new cancerous centre, starting from a primary tumour, in another organor different tissue. This metastasic process includes a series ofsequential stages in which the tumoral cells must interact with thecellular components and the tissues of the host. These stages are asfollows: separation of tumoral cells from a primary tumour; invasion ofintravascular space; migration through the vascular or lymphatic systemto other tissues; adhesion to the vascular endothelium; extravasationand invasion of the new tissue; and formation of the secondary tumour.

Throughout this process, the metastasic tumoral cells interact with thecomponents of the extracellular matrices and, specifically, with thebasal membranes, through their adhesion thereto, provoking theirdeterioration through the action of proteolytic enzymes produced bythemselves and/or by the actual host cells, stimulated by the tumoralcells. Thus, the alteration to the cellular adhesion properties is anindispensable element for the appearance of metastasis, since it is aprocess tied to the liberation of cells from the initial tumour, totheir migration and to their implantation in new tissue.

The principal molecules of adhesion that intervene in this interactionare the integrins. The integrins are a family of transmembraneglycoproteins, formed by two chains a and p joined by non-covalent bonds(hydrophobes). Among other functions, the integrins act a receptors ofdetermined proteins of the extracellular matrix, like the Lamanin, theFibronectin, the Vitronectin and the Collagen (Ruoslahti, E., Giancotti,F. G., Cancer Cells (1989), 1, 4, 119-126). Recently that a change inthe expression of the integrins in the tumoral cells has beendemonstrated whereby their presence in this type of cell is increased(Dedhar, S., Saulmier, R., Cell Biol. (1990), 11, 481-489). Thisincrease is the responsible factor for the adhesion to the extracellularmatrix and for the acquisition of metastasic potential.

The main treatment employed for the elimination of tumours is theadministration of cytostatics by endovenous route, particularly thosebelonging to the anthracycline family (Young, R. C., Ozols, R. F.,Myers, C. E., N. Eng. J. Med. (1981), 305, 139-153). But, due to theirlack of selectivity with respect to the tumoral cells, to thedevelopment of resistance to these drugs by the malignant cells and tothe different response of the primary tumour and of the metastasis withrespect to their action, this type of therapy usually gives rise to theappearance of serious secondary effects, some of which are of a chronicor irreversible nature.

Consequently, the main objective of current chemotherapy centres onachieving an enhanced antitumoral effectiveness and reducing thetoxicity of these drugs. One way of doing this could consist in gettingthe cytostatic, in a suitable concentration, to reach the target cells,resulting in the selective destruction of the primary tumour or themetastases without any healthy cell being affected. In this manner anincreased therapeutic index of the drug would be produced and moreeffective therapies achieved.

In these systems the drug is incorporated in the liposome aqueous spaceswhen it is hydrophilic or it is distributed between these and the lipidbilayers when it has a more lipophilic character. Once the drug isencapsulated, it can be administered to the patient under treatment.

Various researchers have shown that the use of liposomes for theadministration of antineoplastics often enhances the traditional methodsof administration, see, for example: Gabizon et al.: Cancer Res. (1982)42, 4734-4739 and Van Hossel et al.: Cancer Res. (1984) 44, 3698-3705.

It has been observed, by means of the employment of various animalmodels, that the encapsulation of doxorubicin in liposomes reducessignificantly the secondary effects of toxicity, both chronic and acute.See, by way of example, Rahman et al.: Cancer Res. (1980) 40, 1532-1537,Forssen et al.: Proc. Natl. Acad. Sci. USE (1981) 78, 1873-1877, Olsonet al.: Eur. J. Cancer Clin. Oncol. (1982) 18 167-176, Rahman et al.:Cancer Res. (1985) 45, 796-803 and Gabizon et al.: J. Natl. Cancer Inst.(1986) 77, 459-467. Additionally, other toxicity indicators, such asalopecia, weight loss, nausea, vomiting, and also dermal necrosis byextravasation can be reduced in a significant manner with theadministration of doxorubicin in liposomes. Forssen et al.: CancerTreat. Rep. (1983) 67, 481-484; see also the references cited above inthis paragraph.

It has likewise been established in various tumoral models that thissignificant reduction in toxicity is not produced at the expense of adiminution in antitumoral effectiveness. As well as the references citedabove, see Rahman et al.: Chemother. Pharmacol. (1986) 16, 22-27,Gabizon et al.: Cancer Res. (1983) 43, 4730-4735 and Br. J. cancer(1985) 51, 681-689, Mayhew et al.: J. Natl. Cancer Inst. (1987) 78,707-713, Forssen et al.: Cancer Res. (1983) 43, 546-550, and Storm etal.: Cancer Res. (1987) 47, 3366-3372.

Given the incidence and the special characteristics of the cancerousmetastases, antimetastasic therapy is one of the fields in which mosteffort has been applied in the hunt for new alternatives to conventionaltreatments. For their mechanisms of action, their proven higheffectiveness and their high toxicity, the anthracyclines form thefamily of cytostatics most studied in the field of drug encapsulation incontrolled release systems such as the liposomes, which can beappreciated from the growing number of patents that have been appearingunder this heading, of which 80% of the total of those existing forliposomes are applied to cytostatics.

The first generation of liposomes containing anthracyclines correspondedto vesicles formed by PC, PG and cholesterol, in the aqueous interiorspace of which the drug was encapsulated. These liposomes showed adiminution in the toxicity of the drug, though their antitumoralactivity was no greater than that of the free drug, they only improvedthe activity of the drug in the case of tumoral models in which themetastasic cells were spread through the liver, the most accessibleorgan for the liposomes, but not when the tumoral growth is local(Mayhew, E., Rustum, Y., Biol. Cell. (1983), 47, 81-86). In addition,due to their rapid capture by the macrophages of the endoplasmaticreticle, their permanence in the organism after the intravenousinjection was reduced to a few hours. For this reason, and despite theintensive research carried out, that has been no formulation to satisfythe expectations initially placed on liposomes as transporters ofcytostatics. It was during the eighties that the situation changed withthe appearance of the first publications in which liposomes weredescribed that presented glycolipids (Allen, T. M., Hansen, C.,Rutledge, J., Biochem. Biophys. Acta (1989), 981, 27-35; Mori, A.,Klivanov, A. L., Torchilin, V. P., Huang, L., FEBS Lett. (1991), 284,263-266) or hydrophilic polymers like the polyethylene glycol (PEG)(Blume, G., Ceve, C., Biochem. Biophys. Acta (1990), 1029, 91-97, Allen,T. M., Hansen, C., Martin, F., Redemann, C., Yau-Young, A., Biochem.Biophys. Acta (1991), 1066, 29-36) on their surface for the purpose ofaugmenting their time of circulation in the blood stream, obtainingthereby the so-called “second generation liposomes” or “stealthliposomes”. It seems this stabilising effect of the PEG and theglycolipids is due to their hydrophilic properties which preventaggregates being formed on the surface of the liposome and permit it notto be recognised as ligand of any cellular receptor nor of any plasmaticprotein. Moreover, their presence on the surface of the liposomesproduces a steric effect, as it hinders the action of the opsonins andother blood proteins, and reduces the accessibility of the macrophagereceptors to the phosphate groups of the phospholipids, which results inan increased time of circulation in the blood.

Subsequently some authors found that it was possible to improve thestability and effectiveness characteristics of these liposomes throughthe incorporation of additives which inhibited lipid peroxidation likevitamin E acetate, BHT or those derived from chromans (EP-0274174,WO-8500968, WO-9202208 and U.S. Pat. No. 5,605,703).

Despite the fact that these galenic forms offered a series of benefitsover conventional forms, there remains pending the possibility ofsteering the vesicles to target cells in order to improve theeffectiveness with smaller drug doses and of suppressing or reducingsecondary effects.

The basic idea is to incorporate on the surface of the liposome anychemical body capable of being recognised selectively by the targetcells. The success in steering the liposomes toward the target cellsresides in an adequate choice of vector molecule.

The process of selecting any chemical structure capable of steering theliposomes to tumoral cells is by no manner trivial, as specifically inthe case of tumoral cells there exists a great diversity of proteinslocated in the membrane as well as of antigens and surface receptorsthat vary according to the metastasic potential, the proliferatingactivity and the tissue in question, in such a manner that, althoughthey could be used as a base for selecting recognition structures, inpractice the choice is not immediate. Furthermore, in many cases, thetrue situation is that the proliferating and/or tumoral cells have as adifferentiating feature the over-expression of determined structureswith respect to normal cells.

Knowledge accumulated to date indicates that the adhesion processes andthe proteins involved play an essential role in the development of themetastasic process and of the necessary vascularization for cellularproliferation. From among the proteins most involved in thesemechanisms, Laminin has proved to be a good candidate for steeringliposomes, since it has been conclusively shown that its receptors areover-expressed in the tumoral cells.

Laminin is the majority component of the basal membrane of cells aftercollagen. It is a glycoprotein formed by three polypeptide chains: a(440 kDa), β (200 kDa) and γ (220 kDa), which are arranged in the shapeof a cross, with two short arms and one long arm. The bonds between thechains are formed by di-sulphide links and by interactions of thenon-covalent type, forming an asymmetric molecule in which differentstructural domains are located.

The cells have different specific membrane receptors that recognisepeptide sequences and/or functional domains of the molecule of theLaminin.

These receptors can be classified into two groups: integrins andnon-integrins.

The integrins are formed by two trans-membrane polypeptide chains, a andβ, in non-covalent association. These molecules are the receptorsthrough which the cells adhere to the components of the extracellularmatrix. Some of them also intervene in the cell-to-cell recognition.Each one recognises specific peptide sequences which are present in themolecules of the matrix, like for example Laminin.

Among the non-integrin-type receptors, that most studied is the receptorfor 67 kDa for Laminin, and it has been isolated and identified startingfrom various cellular tissues, among which are the carcinomas. Moreover,it has been verified that the metastasic tumoral cells express on theircellular surface more receptors for Laminin than normal cells, for whichreason this receptor could be considered to be a marker of tumoralprogression and an indicator of the aggressivity of many types oftumour.

The Laminin presents various metastasic activities, such as:

-   -   causing cellular adhesion, growth and extension;    -   stimulating the distinction between epithelial and tumoral        cells;    -   provoking cellular migration;    -   facilitating malignancy of tumoral cells by their presence on        the surface, which makes their invasivity and metastasic        activity increase.

Determined studies have shown the different functions of Laminin aremediatized by specific peptide sequences present in the Lamininmolecule, such as: the sequence of five amino-acids SIKVAVS, that isfound located in the fragment PA22-2 of the Laminin α chain.Specifically the most active zone of this region is the pentapeptideSIKVAVS.

SUMMARY OF THE INVENTION

At present, studies on liposomes are directed at their steering towardsor targeting of tumoral cells through incorporation on the surface ofthe ligand vesicles, as may be antibodies, peptides and proteins,capable of recognising and linking specifically with this type of cell(Allen T. M., Austin, G. A., Chonn, A., Lin, L., Lee, K. C., Biochem.Biophys. Acta (1991), 1061, 56-63).

Thus, the object of the present invention consists of a new applicationfor anticancerous drugs encapsulated in liposomes, which present as maincharacteristic their being covered by lipopeptides proceeding from thestructure of the laminins—especially the SIKVAVS sequence—in such amanner that the liposomes so prepared demonstrate a high selectivityregarding tumoral cells and therefore increase the effectiveness of theencapsulated anticancerous drug. MEANING OF THE ABBREVIATIONS USED INTHE INVENTION DXR: Doxorubicin PC: Phosphatidylcholine PL: Phospholipidsof hydrogenated egg PG: Phosphatidylglycerol CHOL: Cholesterol CROM:Chroman-6 A Ala Alanine C Cys Cisteine D Asp Aspartic acid E GluGlutamic acid F Phe Phenylalanine G Gly Glycin H His Histidine I IleIsoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn AsparagineP Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T ThrThreonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine Lam2M:myristoyl-PEGAD Lam9: myristoyl-YESIKVAVS Lam9Cys: myristoyl-CYESIKVAVSLam9Cys-b-Ala: myristoyl-AAAAACYESIKVAVS AG10: GYSRARKEAASIKVAVSARKE(E8)-2-4G: NPWHSIYITRFG mir: myristoyl DOX: Doxorubicin

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related with the preparation and use ofliposomes containing anticancerous drugs.

The liposomes of the present invention have as their main characteristicthat they are coated with fragments of hydrophobically derivatizedpeptides (overlaid lipopeptides) in such a manner that the liposome soprepared offer a high targeting capability with regard to tumoral cells,thereby increasing the effectiveness of the encapsulated anticancerousdrug.

Surprisingly it was shown that the active overlaid lipopeptides in vitroprior to being incorporated in liposomes, would totally lose theirtargeting capability when incorporated in liposomes, for which reason,according to the invention, peptide spacers were developed which,intercalated between the sequence and the lipophile chain of theoverlaid lipopeptide would, strangely, permit the targeting ability ofthe active peptide sequence to be maintained.

In this way the structure of the overlaid lipopeptide (hydrophobicallyderivatized peptide) is as follows: Lipid Fragment Spacer ActiveSequence

Consequently, the object of the present invention resides in thepreparation and use of liposomes containing anticancerous drugs which ontheir surface have peptide fragments derivatized from Laminin (overlaidlipopeptides), made up of the following three structural blocks: a lipidfragment, an active oligopeptide and an oligopeptide spacer among otherfragments.

The lipid fragments are fatty acids of carbonated chain length betweenC6 and C20. More specifically decanoyl, myristoyl and stearoyl.

The spacer fragment consists of oligopeptides inactive with respect toLaminin having a length lying between five and ten amino acid residues.More specifically, with a length of seven to nine amino acid residues,and more specifically the sequences AAAAACYE, SSAAACYE and RKERKECYE.

The active sequence consists of the oligopeptide SIKVAVS.

The liposome-forming lipids are well known. Generally phospholipids areincluded, with net neutral or negative charge, and a sterol, likecholesterol. The choice of the lipids is performed based on therequirements with respect to the final liposome size, to the drug to beencapsulated and to the desired stability for the preparation. Usuallythe largest lipid component of the liposomes is the phosphatidyl choline(PC). The PCs differ from each other in the length and degree ofsaturation of their acylic chains and can isolated from natural orsynthesised sources. The inclusion of a negatively charged phospholipidfavours the stability of the liposome solution and prevents thespontaneous aggregation of the liposomes.

The negatively charged phospholipids most employed are the phosphatidylglycerol (PG), phosphatidyl serine (PS) and the phosphatidyl inositol(PI), among others. The proportion used, of neutral phospholipid tonegatively charged phospholipid ranges from 10:2 to 10:10 respectively.The inclusion of cholesterol generally favours the stability of theliposomes by causing the permeability of the membrane to diminish withrespect to ions and small polar molecules and likewise reduces thepenetration capacity of a series of proteins between the bilayers thatcould result in a greater disorder among these. Typically the proportionof cholesterol used runs from 0 to 50% of total lipids.

Optionally, the liposomes object of the present invention, can containadditives that permit enhancement of their stability properties orreduce the toxicity of the encapsulated drug. For example, mention canbe made of the lipid oxidation inhibitors such as those described in thepatents U.S. Pat. No. 5,605,703, EP 0274174, WO-8500968 and WO 9202208.

The anticancerous drugs that can be encapsulated in the liposomes of thepresent invention include, but are not limited to:

-   Nirogenated mustard analogues like Cyclophosphamide; Melphalan;-   Iphosphamide; or Trophosphamide;-   Ethylenimines like Thiotepa;-   Nitrosoureas like Carmustine;-   Leased agents like Temozolomide; or Dacarbazine;-   Analogous antimetabolites of Folic acid like Methotrexate or    Raltitrexed;-   Analogues of Purines like Thioguanine, Cladribine or Fludarabine;-   Analogues of Pyrimidines like Fluorouracil, Tegafur or Gemcitabine;-   Alkaloids of Vinca and analogues like Vinblastine, Vincristine or    Vinorelbine;-   Derivatives of Podophyllotoxin like Etoposide, Taxanes, Docetaxel or    Paclitaxel;-   Anthracyclines and similar like Doxorubicin, Epirubicin, Idarubicin    and Mitoxantrone;-   Other cytotoxic antibiotics like Bleomycin and Mitomycin;-   Platinum compounds like Cisplatin, Carboplatin and Oxaliplatin;-   Monoclonal antibodies like Rituximab;-   Other antineoplastic agents like Pentostatin, Miltefosine,    Estramustine, Topotecan, Irinotecan and Bicalutamide.

In accordance with that described above, the liposomes of the presentinvention present the following characteristics:

-   -   a) A lipid concentration of 1 and 100 mg/ml, and preferably        around 10 mg/ml.    -   b) The component lipids are phospholipids, of both natural and        synthetic origin, and cholesterol.    -   c) The proportion of cholesterol, with respect to the quantity        of total lipids, is between 0 and 50%, preferably between 35 and        50%.    -   d) The phospholipids present are phosphatidyl choline, which has        no net charge, and optionally another, negatively-charged        phospholipid, preferentially phosphatidyl glycerol.    -   e) The ratio of the neutral phospholipid to that negatively        charged lies between 10:2 and 10:10 and preferably between 10:7        and 10:10 respectively.    -   f) Optionally the liposomes can contain other additives like for        example lipid oxidation inhibitors like those described in the        patents U.S. Pat. No. 5,605,703, EP 0274174, WO-8500968 and        WO-9202208.    -   g) The concentration of peptide would oscillate between 0.1 and        1 mg/ml, and preferably around 0.5 mg/ml.    -   h) The liposomes are formed in an aqueous solution, tamponed or        not, physiologically isotonic. For example, 0.9% NaCl.    -   i) The size of the liposomes shall be in any case less than 500        nm, and preferably less than 300 nm and more specifically        between 50 nm and 250 nm.

Preparation of the Liposomes and Incorporation of the Drug:

A preferred method is that presented by Bangham et al. in whichmultilamellar liposomes (MLVs) are obtained which heterogeneous in size.In this method the forming lipids are dissolved in a suitable organicsolvent that is subsequently removed by rotary evaporation under vacuum.The lipid film formed is subjected to hydration with an adequate aqueousmedium containing the drug, by means of manual or mechanical agitation.The heterogeneous suspension of MLVs is subjected to whatever of theknown procedures for reduction and homogenisation of sizes. For example,two preferred procedures are that of sonication with Titanium probe toobtain SUV liposomes and the extrusion through polycarbonate filters ofthe MLV solution to obtain VET liposomes.

Preparation of the Peptide Fragments:

The sysnthesis of the peptides is carried out using the solid phasemethod of Merrifield (1962) with Fmoc/tBu approach.

Incorporation of Lipopeptide:

The lipopeptides employed were acyl-oligopeptides, being of preferencethe acyl group with linear saturated hydrocarbon chains of length C6 toC20—preferentially the decanoyl, myristoyl or stearoyl. The lipopeptideswere mixed with the rest of the components that were to constitute theliposomes, or else they were incorporated in the liposomes by incubationat 60° C. of these lipopeptides and the vesicles, since, as the bilayerswere in a gel state, permitted the incorporation of the hydrophobic partof these derivatives in their interior. In both cases the hydrophobiczone of the derivatives ought to remain forming the bilayer, whilst thepeptide sequence would remain on the hydrophilic exterior.

By way of illustration, but not restrictively, the procedure detailed inthe present patent is described hereunder by means of several practicalexamples.

EXAMPLE 1 Synthesis of Active Peptides with Carboxylic End

The synthesis of peptides derived from Laminin is carried out followingthe solid phase method of Merrifield (1962) with Fmoc/tBu approach.

In order to obtain a sequence with carboxylic end, as the solidsynthesis support, a Wang resin is employed with a degree offunctionalisation of 0.72 meq/g of resin which was submitted to thetreatment outlined in the table below: TABLE 1 Washing protocol for thepeptidyl resin Step Reagent Repetitions Time 1 DMF 3 times 1 minute 2Dichloromethane 3 times 1 minute 3 Tertiary amyl 3 times 1 minutealcohol 4 Ether Until dry —

In general, the starting point was 1 gram of Wang resin in a syringewith a filter coupled to a vacuum system, and it was dimethylformamide(DMF) was blown in for 30 minutes. In parallel, in a filter weightscale, the necessary amount was weighed of the first Fmoc-amino acid andit was dissolved in DMF, adding to this solution the coupling agents4-dimethyl amino pyridine (4-DMAP) and diisopropylcarbodiimide (DIPCDI)(0.3:1, molar). All the reagents were used in an excess of 5 times withrespect to the quantity required to complete the reaction. Thereafter,this mixture was added to the previously drained resin and left to reactfor 2 hours at room temperature with occasional stirring. After thistime the resin was washed with different solvents until completely dry.

Finally the joint quantity of amino acid was evaluated. In the caseswhere the reaction was incomplete, more reagents were added, in aquantity corresponding to one half the initial quantity employed, andleft to react for a further two hours, repeating thereafter the sameprocess of resin-drying and quantifying of the amino acid incorporated.

The union of the remaining Fmoc-amino acids was carried out throughsuccessive stages of deprotection of the amino group and formation ofthe amide bond.

Thus, for the suppression of the Fmoc amino protector group, thepeptidyl-resin was treated once with DMF/piperidine 20% for a minute,the treatment being repeated a second time for 5 minutes. Afterwards,the piperidine was removed with various washings with DMF and theninhydrin test was carried out to check for the complete elimination ofthe Fmoc group (blue colouring). In some cases the deprotection wasperformed with the reagent 1.8-diazabicyclo [5.4.0.]-undec7-eno (DBU)used in the mixture of DMF/piperidine/DBU (48:2:2, v/v/v) by means of asingle treatment of the resin for 7 minutes. At the end of this time theresin was washed various times with DMF and the ninhydrin test performedagain just as in the previous case.

Once the peptidyl-resin was unprotected, the pertinent Fmoc-amino acidwas added to it and the coupling reagents. Depending on the difficultythe synthesised sequence presented, two different combination ofreagents were used:

HOBt and DIPCDI, in the molar proportion 1:1, with the Fmoc-amino acid.

HOBt, DIEA and 2-(1H-benzotriazol-1-il)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), in the molar proporation 1:2:1.

All the reagents were used in an excess of 2.5 times with respect to thequantity necessary.

In both cases the reaction was left for 1 hour, the conclusion beingcontrolled by means of the ninhydrin test for the disappearance of thefree amino groups (yellow colouring). When the reaction was notcomplete, the mixture was left in contact with the resin for 1 morehour, after which the ninhydrin test was repeated. In the event thatthere were still free amino groups in the resin, the latter was washedseveral times with DMF and the reagents were added again in half thequantity initially employed. On some occasions, and despite the reactionbeing repeated, incomplete couplings were produced. In order to be ableto continue with the synthesis without anomalous chains being formed, itwas necessary to block the incomplete chains by acetylation of the aminogroups that still remained free. To this end, the resin was treated with2 mequivalents of acetic anhydride and 1 mequivalent of 4-DMAP for eachmequivalent of peptidyl-resin during 30 minutes. Next it was washed withDMF and a ninhydrin test was run to check the total disappearance of theamino groups (yellow colouring).

The ninhydrin test was substituted by the chloranil test in the event ofdetecting secondary amino groups of amino acids like proline, since theninhydrin does not react with said groups.

EXAMPLE Sysnthsis of the Peptides with Terminal Amino End

The peptides with carboxamide end are obtained from the resinp-methylbenzhydrylamine (MBHA). This resin needs a special initialtreatment which comprises various washings with an acidic mixture ofDCM/TFA at 40%, being left finally in contact with the resin for 20minutes. Afterwards, to remove the acid, it was washed 5 times with DCMfor 1 minute each time, and to neutralise it, the resin was treated withthe base mixture DCM/diisopropylethylamine (DIEA) at 5%, until it wasfound that the resin pH was base. Finally, to remove the DIEA, it waswashed various times with DCM.

Next, the coupling was carried out of the acidic spacerp-[(R,S)-alpha[1-9H-fluorene-9-e)-methoxyformamide]-2,4-dimethoxybenzyl]-phenoxyacetic (AM), protected with theFmoc group, which is what provides the sequence with its amide end. Forthis, the Fmoc-AM was weighed in an excess of 1.5 times the quantityrequired, and it was added to the resin together with the reagentshydroxybenzotriazole (HOBt) and DIPCDI (1:1, molar), also in excess,leaving the reaction to take place for 90 minutes. The conclusion of thereaction was determined by means of the Kaiser test or ninhydrin test,checking for the disappearance of free amino groups from the resin. Inthe event that all the spacer had not linked, the reaction was repeatedonce more, using half of the initial quanity of reagents employed. Onceall the spacer had linked, the resin was washed various times with DMFin order to remove the reagents in excess.

The coupling of the remaining Fmoc-amino acids was performed throughsuccessive stages of deprotection of the amino group and of amide linkformation, just as described in Example 1.

EXAMPLE 3 Deprotection and De-Anchoring of the Peptide

For the deprotection of the free peptide sequence, the Fmoc: group isfirst removed from the terminal-amino end following the protocol givenin the table below: TABLE 2 Deprotection and de-anchoring protocol ofthe peptide Step Reagent Repetitions Time 1 DMF 3 times 1 minute 2DMF/piperidine 1 time 1 minute 20% 3 DMF/piperidine 3 times 5 minutes20% 4 DMF 3 times 1 minute 5 DCM 3 times 1 minute 6 Tertiary amyl 3times 1 minute alcohol 7 Ether Until dry

In some syntheses the DMF/piperidine at 20% is replaced with the mixtureDMF/piperidine/DBU (48:2:2, v/v/v), which is left in contact with thepeptidyl-resin for 7 minutes.

Next the peptide was de-anchored from the resin and the protector groupsremoved from the amino acid functional chains, in a single step. Toachieve this, various TFA mixtures were prepared with differentscavengers like anisol, thioanisole, phenol, mercaptoethanol, and water,according to the protector groups present in the peptide chains. Analiquot was weighed of the peptide-resin a syringe with filter coupledto a vacuum system and the acidic mixture of scavengers added to it,being left in contact with the resin for 2 to 3 hours, at roomtemperature with occasional stirring. After this time elapsed, resin wasfiltered and washed 3 times with TFA, the filtrates and the washingproducts being collected in a tube. First the TFA was evaporated offwith Nitrogen and afterwards cold diethyl ether was added, obtaining awhite precipitate (free peptide). The precipitate was centrifuged at3000 rpm for 15 minutes, the supernatant being drained off and theprocess repeated 5 more times. Finally the traces of ether were removedfrom the solid with Nitrogen, it was re-dissolved in water or acetic at10%, depending on the peptide solubility, and lyophilised to obtain theraw free peptide product completely dry.

EXAMPLE 4 Hydrophobic Derivatization of the Peptide Sequences in SolidPhase

The fatty acids were coupled to the sequences in the same form as theFmoc-amino acids, by means of the formation of an amide bond with thecarboxylic group of the fatty acid.

Thus, an aliquot was weighed of the peptidyl-resin in a syringe withfilter attached to a vacuum pump and was swollen with DMF. Next thedeprotection of the Fmoc group was carried out. Once deprotected, thefatty acid employed in each case was added, in an excess of 2.5 times,together with the systhesis reagents DPCDI/HOBt or, TBTU/DIEA/HOBt,depending on the peptide sequence in question. The conclusion of thereaction was determined, just as during the synthesis, by the ninhydrintest for disappearance of free amine groups.

To obtain the free hydrophobic derivative, the peptidyl-resin wastreated with the same acidic mixture of TFA and scavengers, and underidentical conditions to those employed in the de-anchoring of theinitial peptide sequence.

EXAMPLE 5 Obtaining Liposomes Containing Doxorubicin and a Lipopeptidecovering the surface of the liposome

Initially, and in all cases, large multilamellar liposomes (MLV) wereprepared following the method described by Bangham. From these, and bysonication, the small unilamellar liposomes (SUV) were obtained.

All the material and the solutions employed were sterile and, during thewhole process, the work was carried out under a laminar flow hood tomaintain sterility.

The liposomes prepared with the hydrophobic derivatives of the twoactive sequences had in their composition: phosphatidyl choline (PC),phosphatidyl glycerol (PG), cholesterol and Chroman-6. To obtain themthe following procedure was adopted:

Thus, in the first place, SUV liposomes were prepared. The PC, PG,cholesterol and the Chroman-6 were weighed, and dissolved in Chloroform,the solvent being evaporated off in the rotary evaporator in order toform a lipid film. Any traces of solvent that might remain were removedby lyophilisation lasting 1 hour.

After this period had elapsed, the film was hydrated with 1 mL of NaClat 0.9%, maintaining the ball in a bath at 60 degrees Celsius for 1hour. To the MLV liposomes obtained, 1.2 mL of a Doxorubicin solutionwas added having a concentration equal to 2 mg/mL (2.4 mg). Thepreparation was left in repose for 15 minutes in a bath at 60 degreesCelsius and afterwards the ball was kept in a vacuum-free rotaryevaporator which turned slowly for a period of 20 minutes.

In order to obtain SUV liposomes, the MLV were subjected to sonicationin an ultrasonic bath for 8 cycles each lasting 2 minutes, separated by5-minute intervals of repose in a bath at 60 degrees Celsius.

The incorporation of the lipopeptides was carried out by mixing analiquot of 200 μL of liposomes, 200 μL of NaCl at 0.9% and 12 μL of asolution of lipopeptide in DMSO (c=10 mg/mL). The mixture was left inrepose at 60 degrees Celsius for one hour and afterwards at roomtemperature for a further 30 minutes.

Alternatively, the liposomes were prepared incorporating the lipopeptidefrom the beginning. Thus, the PC, PG, Cholesterol and Chroman-6 lipidswere mixed with an aliquot of the lipopeptide dissolved inchloroform/methanol, in the same molar ratio as in the previous case.The rest of the procedure is identical to the previous case.

Finally, to remove the Doxorubicin not encalsulated and the lipopeptidenot incorporated, the sample was placed in a PD-10 column (SephadexG-25). For this, the column was first balanced with NaCl at 0.9%. Oncebalanced, the sample was added, which was also eluted with NaCl at 0.9%,until it overflowed from the column. The volume of liposomes obtainedwas made up to 2 mL.

Following this process, the following types of liposomes were preparedincorporating Doxorubicin: Lipid Conc. of Conc. of Coating Conc. ofLiposome composition Lipids drug lipopeptide peptide sizePC/PG/Chol./Chr.  9.91 mg/mL 1.04 mg/mL Myristic-(A)₅- 0.42 mg/mL 160 nmCYESIKVAVS PC/PG/Chol./Chr. 14.05 mg/mL  1.5 mg/mL Myristic-  1.1 mg/mL115 nm PEAGD

EXAMPLE 6 Obtaining Liposomes Containing Paclitaxel and a LipopeptideCoating the the Surface of the Liposome

Initially, and in all cases, large multilamellar liposomes (MLV) wereprepared following the method described by Bangham. From these, and bysonication, small unilamellar liposomes were obtained.

All the material and the solutions employed were sterile and, during thewhole process, the work was carried out under a laminar flow hood tomaintain sterility.

The liposomes prepared with the hydrophobic derivatives of the activesequences had in their composition: phosphatidyl choline (PC),phosphatidyl glycerol (PG) and cholesterol. To obtain them the followingprocedure was adopted:

Thus, in the first place, SUV liposomes were prepared. The PC, andcholesterol were weighed, and dissolved in Chloroform, the solvent beingevaporated off in the rotary evaporator in order to form a lipid film.Any traces of solvent that might remain were removed by lyophilisationlasting 1 hour.

After this period had elapsed, the film was hydrated with 1 mL of NaClat 0.9%, maintaining the ball in a bath at 60 degrees Celsius for 1hour. To the MLV liposomes obtained, 1.2 mL of a Paclitaxel solution wasadded having a concentration equal to 0.5 mg/mL (0.6 mg). Thepreparation was left in repose for 15 minutes in a bath at 60 degreesCelsius and afterwards the ball was kept in a vacuum-free rotaryevaporator which turned slowly for a period of 20 minutes.

In order to obtain SUV liposomes, the MLV were subjected to sonicationin an ultrasonic bath for 8 cycles each lasting 2 minutes, separated by5-minute intervals of repose in a bath at 60 degrees Celsius.

The incorporation of the lipopeptides was carried out by mixing analiquot of 200 μL of liposomes, 200 μL of NaCl at 0.9% and 12 μL of asolution of lipopeptide in DMSO (c=10 mg/mL). The mixture was left inrepose at 60 degrees Celsius for one hour and afterwards at roomtemperature for a further 30 minutes.

Alternatively, the liposomes were prepared incorporating the lipopeptidefrom the beginning. Thus, the PC, PG and Cholesterol lipids were mixedwith an aliquot of the lipopeptide dissolved in chloroform/methanol, inthe same molar ratio as in the previous case. The rest of the procedureis identical to the previous case.

Finally, to remove the Paclitaxel not encapsulated and the lipopeptidenot incorporated, the sample was placed in a PD-10 column (SephadexG-25). For this, the column was first balanced with NaCl at 0.9%. Oncebalanced, the sample was added, which was also eluted with NaCl at 0.9%,until it overflowed from the column. The volume of liposomes obtainedwas made up to 2 mL.

Following this process, the following types of liposomes were preparedincorporating Paclitaxel: Lipid Conc. of Conc. of Coating Conc. ofLiposome composition Lipids drug lipopeptide peptide size PC/PG/Chol.8.93 mg/mL 0.26 mg/mL Myristic-(A)₅- 0.42 mg/mL 140 nm CYESIKVAVSPC/PG/Chol. 13.5 mg/mL  0.4 mg/mL Myristic-  1.1 mg/mL 105 nm PEAGD

EXAMPLE 7 Cellular Adhesion Tests

Solutions of Laminin-1 and synthetic peptides (50 mg/well) were fixed inwells of the 96-well tissue culture plate of TPP (Switzerland). Thewells were dried at room temperature during the night. Before usingthem, the wells were washed with tamponed saline solution free fromCalcium and Magnesium ions. The remaining free radicals of thepolystyrene were blocked by using a 1% BSA solution.

They were cultivated and marked with ⁵¹Cr cells of human fibrosarcomaHT1080. The marked cells were placed (1 cpm/well) in the wells whichcontained the Laminin and the synthetic peptides.

After 30 minutes of incubation at 37 degrees Celsius, the unadheredcells were removed by washing. The adhered cells were smoothed and theradioactivity measured. The specific percentages of adhesion encounteredare shown in the attached FIG. 1.

EXAMPLE 8 Inhibition of Cellular Adhesion to Laminin (Complete Molecule)in Vitro by Peptides of the Laminin

Following the procedure described under Example 1 HT-1080 cells markedwith ⁵¹Cr were adhered, in wells (0.32 cm²) coated with 1 μg of Laminin.The adhered cells were incubated with different concentrations of ofsynthetic peptide fragments of Laminins. The results obtained are shownin the attached FIG. 2.

EXAMPLE 9 Anti-Proliferative Effect of Doxorubicin Liposomes DirectedAgainst Specific Receptors of Laminin Peptides in Tumoral Cells

The anti-proliferative effect of Doxorubicin was analysed by followingthe MTT method. HT1080 cells obtained from exponential cultures weresown in 0.36 cm² wells (96-well tissue culture plates of TPP,Switzerland) with a density of 5000 cells per well. One day later, thecells were washed and incubated for two hours with liposomes containingDoxorubicin. The different liposome formulations were adapted to thesame drug concentration and the test was carried out in parallel wells(increasing the concentration of Doxorubicin from 0.01 μg/ml to 10μg/ml. After the incubation, the cells were washed five times with PBSand incubated for three days in a complete medium. After this period, toeach well was added 50 μL of PBS containing 1 mg/ml of MTT (tetrazoliumsalt, Sigma) and they were incubated for a further four hours. Theintracellular crystals of Fromazan resulting from the reduction of thetetrazolium salt, only present in the active cells, were dissolved inDMSO. The number of metabolically active cells was estimated bymeasuring the absorbance of this solution of DMSO at 540 nm.

The percentage of cytostatic activity was calculated according to theformula (A−B)/A×100, where A is the absorbance in tumoral cellsincubated in a control medium and B is the absorbance in tumoral cellsincubated with the liposome preparations.

The results of the resulting cytostasis are shown in the attached FIG.3, in which:

-   -   the results present the mean+/−the standard desiccation of three        independent experiments performed in triplicate;    -   The IC₅₀ is defined as the drug concentration at which 50% of        the cells survive in comparison with the control lot; and    -   P>0.05; Student test t.

EXAMPLE 10 Biodistribution of Doxorubicin Administered as Free Drug orLiposome Preparation (PC/PG/Chol/myristoyl-AAAAACYESIKVAVS)/Doxorubicin)in Tumour-Bearing Animals

Animals: The tests were performed on naked and immuno-suppressed BALB/cmice obtained for the animal production area of IFFA CREDO Inc. (Lyons,France). The animals were kept in laminar flow cabins in pathogen-freeconditions and were used when they reached an age of 8 weeks.

Cellular culture conditions: Cells of HT1080 human fibrosarcoma weremade to grow in Ham's F-12 medium (GIBCO, Grand Island, N.Y.)supplemented with 10% of bovine fetal serum, Sodium pyruvate,non-essential amino acids, L-glutamine, and vitamin solution (GIBCO,Grand Island, N.Y.). The cultures were kept in plastic and incubated in5% CO₂-95% air at 37 degrees Celsius in humidified incubators. Thecellular line was examined to certify the absence of Mycoplasma.

The tumoral cells were harvested from the sub-confluent cultures (50-70%confluence) by treating with trypsin (0.25%) and EDITA (0.02%). Thecells were washed in a supplemented medium and afterwards werere-suspended in a Hank Balanced Saline Solution (HBSS) for theirsubsequent injection. Only monocellular suspensions with a viability ofmore than 90% (determined by colouring with Trypan blue) were used forthe in vivo studies.

Biodistribution test: HT-1080 cells at a concentration of 1×10⁷ cells/mLof HBSS were pre-mixed with an equal volume of liquid Matrigel(Collaborative Biomedical Products, Bedford, Mass.) 10 mg/mL. Of theresulting suspension, 0.02 mL were inoculated subcutaneously into theleft-hand flank of the mice. Tumour growth was monitored twice weekly.When the tumours attained a volume of 1 cm³ (day 25 after injection ofthe cells), the mice received a single intravenous dose of Doxorubicin(5 mg/kg) in liposome preparation or free drug form. At times of 30minutes, 5 hours and 24 hours from the administration of the drug, themice were sacrificed and samples were taken of tumoral tissue andplasma. The results obtained are shown in FIGS. 4 and 5 attached.

1. Liposomes encapsulating anticancerous drugs, characterised in thatthey are coated with a lipopeptide composed of three substructures (FIG.3): a lipid fragment, an active oligopeptide and an oligopeptide spacerbetween the other two fragments.
 2. Liposomes encapsulatinganticancerous drugs, in accordance with claim 1, characterised in thatthe lipid fragment of the coating lipopeptide are fatty acids of carbonchain length between C6 and C20.
 3. Liposomes encapsulatinganticancerous drugs, in accordance with claims 1 and 2, characterised inthat the lipid fragment of the coating lipopeptide is preferablydecanoyl, myristoyl or stearoyl.
 4. Liposomes encapsulatinganticancerous drugs, in accordance with claims 1 to 3, characterised inthat the active sequence fragment of the coating lipopeptide is SIKVAVS.5. Liposomes encapsulating anticancerous drugs, in accordance withclaims 1 to 4, characterised in that the oligopeptide spacer between theactive sequence fragment and the lipid fragment of the coatinglipopeptide is an oligopeptide having a length between five and tenamino acid residues.
 6. Liposomes encapsulating anticancerous drugs, inaccordance with claims 1 to 5, characterised in that the oligopeptidespacer between the active sequence fragment and the lipid fragment ofthe coating lipopeptide is one of the following sequences: AAAAACYE,SSAAACYE or RKERKECYE.
 7. Liposomes encapsulating anticancerous drugs,in accordance with claims 1 to 6, characterised in that the ratio oftotal lipids forming liposomes to drug lies between 20:1 and 2:1, andpreferably 10:1.
 8. Liposomes encapsulating anticancerous drugs, inaccordance with claims 1 to 7, characterised in that the lipidscomponents of the liposomes are phospholipids, of both natural andsynthetic origin, and cholesterol.
 9. Liposomes encapsulatinganticancerous drugs, in accordance with claims 1 to 8, characterised inthat the phospholipids present are preferentially phosphatidylcholine,which has no net charge, and another phospholipid charged negatively,preferentially phosphatidylglycerol.
 10. Liposomes encapsulatinganticancerous drugs, in accordance with claims 1 to 9, characterised inthat the ratio of the neutral phospholipid to that charged negativelylies between 10:2 and 10:10, preferentially between 10:7 and 10:10respectively.
 11. Liposomes encapsulating anticancerous drugs, inaccordance with claims 1 to 10, characterised in that the proportion ofcholesterol, with respect to the total amount of lipids, lies between 0and 50% and preferentially between 35 and 50%.
 12. Liposomesencapsulating anticancerous drugs, in accordance with claims 1 to 11,characterised in that optionally they can contain an inhibition additiveof the lipid peroxidation.
 13. Liposomes encapsulating anticancerousdrugs, in accordance with claims 1 to 12, characterised in that theinhibition additives of the lipid peroxidation are Vitamins and theirderivatives like Vitamin E or Vitamin E acetate, an antioxidantauthorised for pharmaceutical use like BHT or a chroman or chromen, suchas 3,4-dihydride-2,2-dimethyl-6-hydroxy-7-methoxy-2H-1-benzopyrane. 14.Liposomes encapsulating anticancerous drugs, in accordance with claims 1to 13, characterised in that the proportion of coating lipopeptide withrespect to the total amount of lipids, lies between 0.1% and 30%,preferentially between 1% and 15%.
 15. Liposomes encapsulatinganticancerous drugs, in accordance with claims 1 to 14, characterised inthat they have an average size lying between 50 nm and 250 nm. 16.Liposomes in accordance with claims 1 to 15, characterised in that theencapsulating anticancerous drugs are: Nirogenated mustard analogueslike Cyclophosphamide; Melphalan; Iphosphamide; or Trophosphamide;Ethylenimines like Thiotepa; Nitrosoureas like Carmustine; Leased agentslike Temozolomide; or Dacarbazine; Analogous antimetabolites of Folicacid like Methotrexate or Raltitrexed; Analogues of Purines likeThioguanine, Cladribine or Fludarabine; Analogues of Pyrimidines likeFluorouracil, Tegafur or Gemcitabine; Alkaloids of Vinca and analogueslike Vinblastine, Vincristine or Vinorelbine; Derivatives ofPodophyllotoxin like Etoposide, Taxanes, Docetaxel or Paclitaxel;Anthracyclines and similar like Doxorubicin, Epirubicin, Idarubicin andMitoxantrone; Other cytotoxic antibiotics like Bleomycin and Mitomycin;Platinum compounds like Cisplatin, Carboplatin and Oxaliplatin;Monoclonal antibodies like Rituximab; Other antineoplastic agents likePentostatin, Miltefosine, Estramustine, Topotecan, Irinotecan andBicalutamide.
 17. Use of the liposomes encapsulating anticancerousdrugs, in accordance with claims 1 to 16, for the intravenousadministration in humans or other mammals for the treatment of malignanttumours.